Gas-to-liquid mass transfer of hydrogen (H2) was investigated in a gas–liquid reactor with a continuous gas phase, a batch liquid phase, and liquid mixing regimes relevant to assessing kinetics of microbial H2 consumption. H2 transfer was quantified in real-time with a H2 microsensor for no mixing, moderate mixing [100 rotations per minute (rpm)], and rapid mixing (200 rpm). The experimental results were simulated by mathematical models to find best-fit values of volumetric mass transfer coefficients—kLa—for H2, which were 1.6/day for no mixing, 7/day for 100 rpm, and 30/day for 200 rpm. Microbiological H2-consumption experiments were conducted with Methanobacterium bryantii M.o.H. to assess effects of H2 mass transfer on microbiological H2-threshold studies. The results illustrate that slow mixing reduced the gas-to-liquid H2 transfer rate, which fell behind the rate of microbiological H2 consumption in the liquid phase. As a result, the liquid-phase H2 concentration remained much lower than the liquid-phase H2 concentration that would be in equilibrium with the gas-phase H2 concentration. Direct measurements of the liquid-phase H2 concentration by an in situ probe demonstrated the problems associated with slow H2 transfer in past H2 threshold studies. The findings indicate that some of the previously reported H2-thresholds most likely were over-estimates due to slow gas-to-liquid H2 transfer. Essential requirements to conduct microbiological H2 threshold experiments are to have vigorous mixing, large gas-to-liquid volume, large interfacial area, and low initial biomass concentration.